Project Summary DNA mismatch repair (MMR) is a post-replicative system of proteins that corrects rare mistakes in the genome of all organisms. In the human genome of 6 billion bases, there are ~ 600 errors per round of replication, per cell. If left uncorrected, errors accumulate as permanent mutations in a genome, and can lead to a disease state in the organism. MutS and MutL homologs are tasked with recognizing a mismatch in 107 correctly paired bases, discriminating between parent and daughter strand, then initiating repair. Single amino acid mutations in MutS and MutL proteins have been linked to hereditary and sporadic colorectal cancer, the third most common cancer worldwide. Although these mutations, mostly associated with MutL, have been identified in cancer cases, it is unclear how MMR deficiencies initiate and advance the disease. Failures in the mismatch repair pathway likely initiate tumorigenesis, but we lack a fundamental understanding of the MMR process. On the molecular level, we know that MutS initially recognizes a DNA mismatch, and undergoes ATP- dependent conformational changes to slide along the DNA. MutL is recruited to the site, and interacts with MutS on DNA to coordinate repair with PCNA, EXO1, DNA polymerase, RFC clamp loader, RPA single strand binding protein, and DNA ligase. We also know that MutL undergoes conformational changes upon ATP binding and hydrolysis, which likely functions to coordinate transient interactions with repair machinery. Previous studies show four distinct conformations of MutL that we believe must be regulated and functional in MMR. MutL is the central player in the middle of the pathway that directs multiple molecular interactions, but how it carries out its functions remains poorly understood. MutL mutations are associated with a spectrum of cancers, thus we need to understand its dynamic molecular interactions and MMR functions, which begin with the MutS-DNA recognition complex. Single molecule fluorescence resonance energy transfer (smFRET) is uniquely capable of investigating the molecular mechanism of MMR that involves multiple transient protein- protein and protein-DNA interactions. The molecular mechanism of mismatch repair is critical for further revealing how mutants fail to repair, and will provide a basis for identifying therapeutic strategies. We hypothesize that mutations in functional ATPase and interfacial regions of MutL are inadequate in their functional conformational changes and fail to advance repair. To explore these open questions, we propose the following specific aims: Specific Aim 1: Characterize the nucleotide-dependent dynamics of MutL conformations in the absence of mismatch DNA in vitro using single molecule FRET. Specific Aim 2: Investigate the dynamics of wild-type and mutant MutL conformations in the context of mismatch repair initiation with nucleotides, MutS, and mismatch DNA in vitro with smFRET.